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Conditional expression of apical membrane antigen 1 in Plasmodium falciparum shows it is required for erythrocyte invasion by merozoites.

Yap A, Azevedo MF, Gilson PR, Weiss GE, O'Neill MT, Wilson DW, Crabb BS, Cowman AF - Cell. Microbiol. (2014)

Bottom Line: DiCre-mediated excision of the loxP-flanked Pfama1 gene results in approximately 80% decreased expression of the protein within one intraerythrocytic growth cycle.This reduces growth by 40%, due to decreased invasion efficiency characterized by a post-invasion defect in sealing of the parasitophorous vacuole.These results show that PfAMA1 is an essential protein for merozoite invasion in P.  falciparum and either directly or indirectly plays a role in resealing of the red blood cell at the posterior end of the invasion event.

View Article: PubMed Central - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., 3052, Australia.

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W2mef/AMA1-loxP and W2mef/AMA1-loxP/DiCre parasites are complemented with the 3D7 AMA1 protein.A. Southern blot indicates that two copies of pAMA1-loxP have integrated into the W2mef Pfama1 locus leading to a duplication of the heterologous 3D7 Pfama1 gene. The 4 and 3 kb AflIII fragments indicate the first and second copy of the full-length integrated plasmid (see Fig. 1). Both 4 and 3 kb signals have equal intensity on the Southern blot, indicating that copy numbers are identical (i.e. 1:1 ratio). The 1.45 kb signal signifies the 3′ end of the modified locus where the second plasmid re-joins the endogenous, disrupted W2mef Pfama1 gene. This signal is a single copy fragment hence it is used as DNA loading control for each track.B. Southern blotting using XhoI restriction enzyme confirms the presence of the 9.3 kb full-length pDiCre plasmid in W2mef/AMA1-loxP/DiCre parasites.C. Immunoblot analysis of late-schizont extracts probed with the specific anti-3D7 AMA1 mAb 3A2 showing the two most abundant forms of AMA1: the 83 kilodalton (kDa) precursor, AMA183 and the 66 kDa processed form, AMA166. The result shows that complementation with 3D7 AMA1 protein has occurred in W2mef/AMA1-loxP and W2mef/AMA1-loxP/DiCre clonal lines (2F9 and B4).D. A polyclonal PfAMA1 antibody detected PfAMA1 signals from all tracks including parental W2mef confirming the result of the previous immunoblot.E. Parental and transgenic parasites were grown in media +/− R1 peptide. R1 peptide binds specifically to 3D7 PfAMA1 but not W2mef PfAMA1. Complementation of W2mef/AMA1-loxP/DiCre parasites with a functional 3D7 PfAMA1 protein is evident as R1 peptide significantly inhibited growth of 3D7, 2F9 and B4 parasites but not W2mef (n = 2 experiments with each done in triplicate). Error bars indicate standard deviation (SD).
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fig02: W2mef/AMA1-loxP and W2mef/AMA1-loxP/DiCre parasites are complemented with the 3D7 AMA1 protein.A. Southern blot indicates that two copies of pAMA1-loxP have integrated into the W2mef Pfama1 locus leading to a duplication of the heterologous 3D7 Pfama1 gene. The 4 and 3 kb AflIII fragments indicate the first and second copy of the full-length integrated plasmid (see Fig. 1). Both 4 and 3 kb signals have equal intensity on the Southern blot, indicating that copy numbers are identical (i.e. 1:1 ratio). The 1.45 kb signal signifies the 3′ end of the modified locus where the second plasmid re-joins the endogenous, disrupted W2mef Pfama1 gene. This signal is a single copy fragment hence it is used as DNA loading control for each track.B. Southern blotting using XhoI restriction enzyme confirms the presence of the 9.3 kb full-length pDiCre plasmid in W2mef/AMA1-loxP/DiCre parasites.C. Immunoblot analysis of late-schizont extracts probed with the specific anti-3D7 AMA1 mAb 3A2 showing the two most abundant forms of AMA1: the 83 kilodalton (kDa) precursor, AMA183 and the 66 kDa processed form, AMA166. The result shows that complementation with 3D7 AMA1 protein has occurred in W2mef/AMA1-loxP and W2mef/AMA1-loxP/DiCre clonal lines (2F9 and B4).D. A polyclonal PfAMA1 antibody detected PfAMA1 signals from all tracks including parental W2mef confirming the result of the previous immunoblot.E. Parental and transgenic parasites were grown in media +/− R1 peptide. R1 peptide binds specifically to 3D7 PfAMA1 but not W2mef PfAMA1. Complementation of W2mef/AMA1-loxP/DiCre parasites with a functional 3D7 PfAMA1 protein is evident as R1 peptide significantly inhibited growth of 3D7, 2F9 and B4 parasites but not W2mef (n = 2 experiments with each done in triplicate). Error bars indicate standard deviation (SD).

Mentions: The gene encoding PfAMA1 is refractory to genetic ablation via conventional knockout strategies (Triglia et al., 2000) and consequently we constructed a P. falciparum line in which it was placed under conditional regulation. To do this we simultaneously disrupted the endogenous Pfama1 gene, in the W2mef line by single recombination, with the plasmid pAMA1-loxP allowing complementation with a transfected gene (encodes the antigenically distinct 3D7 allele) flanked with loxP sites (Fig. 1). The resulting W2mef/AMA1-loxP parasites were cloned by limiting dilution and the clones derived had two copies of the plasmid inserted into the Pfama1 gene resulting in two functional copies of the 3D7 allele (Figs 1 and 2A, Fig. S1).


Conditional expression of apical membrane antigen 1 in Plasmodium falciparum shows it is required for erythrocyte invasion by merozoites.

Yap A, Azevedo MF, Gilson PR, Weiss GE, O'Neill MT, Wilson DW, Crabb BS, Cowman AF - Cell. Microbiol. (2014)

W2mef/AMA1-loxP and W2mef/AMA1-loxP/DiCre parasites are complemented with the 3D7 AMA1 protein.A. Southern blot indicates that two copies of pAMA1-loxP have integrated into the W2mef Pfama1 locus leading to a duplication of the heterologous 3D7 Pfama1 gene. The 4 and 3 kb AflIII fragments indicate the first and second copy of the full-length integrated plasmid (see Fig. 1). Both 4 and 3 kb signals have equal intensity on the Southern blot, indicating that copy numbers are identical (i.e. 1:1 ratio). The 1.45 kb signal signifies the 3′ end of the modified locus where the second plasmid re-joins the endogenous, disrupted W2mef Pfama1 gene. This signal is a single copy fragment hence it is used as DNA loading control for each track.B. Southern blotting using XhoI restriction enzyme confirms the presence of the 9.3 kb full-length pDiCre plasmid in W2mef/AMA1-loxP/DiCre parasites.C. Immunoblot analysis of late-schizont extracts probed with the specific anti-3D7 AMA1 mAb 3A2 showing the two most abundant forms of AMA1: the 83 kilodalton (kDa) precursor, AMA183 and the 66 kDa processed form, AMA166. The result shows that complementation with 3D7 AMA1 protein has occurred in W2mef/AMA1-loxP and W2mef/AMA1-loxP/DiCre clonal lines (2F9 and B4).D. A polyclonal PfAMA1 antibody detected PfAMA1 signals from all tracks including parental W2mef confirming the result of the previous immunoblot.E. Parental and transgenic parasites were grown in media +/− R1 peptide. R1 peptide binds specifically to 3D7 PfAMA1 but not W2mef PfAMA1. Complementation of W2mef/AMA1-loxP/DiCre parasites with a functional 3D7 PfAMA1 protein is evident as R1 peptide significantly inhibited growth of 3D7, 2F9 and B4 parasites but not W2mef (n = 2 experiments with each done in triplicate). Error bars indicate standard deviation (SD).
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fig02: W2mef/AMA1-loxP and W2mef/AMA1-loxP/DiCre parasites are complemented with the 3D7 AMA1 protein.A. Southern blot indicates that two copies of pAMA1-loxP have integrated into the W2mef Pfama1 locus leading to a duplication of the heterologous 3D7 Pfama1 gene. The 4 and 3 kb AflIII fragments indicate the first and second copy of the full-length integrated plasmid (see Fig. 1). Both 4 and 3 kb signals have equal intensity on the Southern blot, indicating that copy numbers are identical (i.e. 1:1 ratio). The 1.45 kb signal signifies the 3′ end of the modified locus where the second plasmid re-joins the endogenous, disrupted W2mef Pfama1 gene. This signal is a single copy fragment hence it is used as DNA loading control for each track.B. Southern blotting using XhoI restriction enzyme confirms the presence of the 9.3 kb full-length pDiCre plasmid in W2mef/AMA1-loxP/DiCre parasites.C. Immunoblot analysis of late-schizont extracts probed with the specific anti-3D7 AMA1 mAb 3A2 showing the two most abundant forms of AMA1: the 83 kilodalton (kDa) precursor, AMA183 and the 66 kDa processed form, AMA166. The result shows that complementation with 3D7 AMA1 protein has occurred in W2mef/AMA1-loxP and W2mef/AMA1-loxP/DiCre clonal lines (2F9 and B4).D. A polyclonal PfAMA1 antibody detected PfAMA1 signals from all tracks including parental W2mef confirming the result of the previous immunoblot.E. Parental and transgenic parasites were grown in media +/− R1 peptide. R1 peptide binds specifically to 3D7 PfAMA1 but not W2mef PfAMA1. Complementation of W2mef/AMA1-loxP/DiCre parasites with a functional 3D7 PfAMA1 protein is evident as R1 peptide significantly inhibited growth of 3D7, 2F9 and B4 parasites but not W2mef (n = 2 experiments with each done in triplicate). Error bars indicate standard deviation (SD).
Mentions: The gene encoding PfAMA1 is refractory to genetic ablation via conventional knockout strategies (Triglia et al., 2000) and consequently we constructed a P. falciparum line in which it was placed under conditional regulation. To do this we simultaneously disrupted the endogenous Pfama1 gene, in the W2mef line by single recombination, with the plasmid pAMA1-loxP allowing complementation with a transfected gene (encodes the antigenically distinct 3D7 allele) flanked with loxP sites (Fig. 1). The resulting W2mef/AMA1-loxP parasites were cloned by limiting dilution and the clones derived had two copies of the plasmid inserted into the Pfama1 gene resulting in two functional copies of the 3D7 allele (Figs 1 and 2A, Fig. S1).

Bottom Line: DiCre-mediated excision of the loxP-flanked Pfama1 gene results in approximately 80% decreased expression of the protein within one intraerythrocytic growth cycle.This reduces growth by 40%, due to decreased invasion efficiency characterized by a post-invasion defect in sealing of the parasitophorous vacuole.These results show that PfAMA1 is an essential protein for merozoite invasion in P.  falciparum and either directly or indirectly plays a role in resealing of the red blood cell at the posterior end of the invasion event.

View Article: PubMed Central - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., 3052, Australia.

Show MeSH
Related in: MedlinePlus